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Prion generation in vitro: amyloid of Ure2p is infectious

Andreas Brachmann1, Ulrich Baxa, Reed Brendon Wickner

  • 1Laboratory of Biochemistry and Genetics, National Institute of Diabetes and Digestive and Kidney Diseases, Bethesda, MD 20892-0830, USA.

The EMBO Journal
|August 13, 2005
PubMed

Insights

Yeast prion [URE3], caused by Ure2 protein, is infectious. Amyloid filaments of recombinant Ure2p are nearly as infectious as cell extracts, confirming their role in prion propagation.

Area of Science:

  • Biochemistry
  • Molecular Biology
  • Yeast Genetics

Background:

  • The prion [URE3] in yeast is associated with the Ure2 protein, but direct evidence linking in vitro formed amyloid filaments to infectivity is lacking.
  • Understanding the structural basis of prion infectivity is crucial for deciphering protein misfolding diseases.

Purpose of the Study:

  • To investigate whether amyloid filaments of recombinant Ure2p can infect yeast cells and serve as the infectious form of the [URE3] prion.
  • To characterize the infectious properties and structural determinants of the [URE3] prion.

Main Methods:

  • Recombinant Ure2 protein was converted into amyloid filaments in vitro.
  • These amyloid filaments were used to infect yeast cells lacking the prion.
  • Infectivity was assessed by transmission to uninfected cells and characterization of [URE3] variants.
  • Size exclusion chromatography and other methods were used to determine the size of infectious particles.

Main Results:

  • Recombinant Ure2p amyloid successfully infected yeast cells, producing various [URE3] variants.
  • Amyloid filaments of Ure2p showed infectivity comparable to cell extracts on a mass basis.
  • The N-terminal 65 amino acids of Ure2p were identified as the determinant of infectivity and [URE3] variants.
  • Infectious particles in vitro and in vivo were consistently larger than 20 nm, consistent with amyloid filaments.

Conclusions:

  • Amyloid filaments of recombinant Ure2p are the infectious form of the [URE3] prion.
  • There is no significant difference between Ure2p amyloid filaments formed in vivo and in vitro.
  • The N-terminal region of Ure2p is critical for [URE3] prion infectivity.

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